CSF findings in patients with voltage gated potassium channel antibody associated limbic encephalitis.
Recently, a new subtype of limbic encephalitis (LE) has been described, serologically characterized by the presence of antibodies against voltage gated potassium channels (VGKC, to be called VGKC-LE). Only little is known about CSF findings in this new disorder. Here we report the results of 29 lumbar punctures in 17 patients with VGKC-LE. Slight pleocytosis, mainly consisting of lymphocytes and monocytes, and elevated total protein concentrations were present in 41 and 47%, respectively. Intrathecal immunoglobulin (Ig) synthesis as defined by the presence of CSF-specific oligoclonal IgG bands, an increased IgG index, or an elevated IgG, IgA, or IgM ratio, was not detected in any of the patients, but dysfunction of the blood-CSF barrier was found in 35%. CSF findings were normal in 23%. Unlike paraneoplastic LE, VGKC-LE is not frequently associated with intrathecal Ig production or markedly elevated white cell counts. Thus, normal CSF findings do not preclude the disease. VGKC-Ab should, therefore, be determined whenever LE is clinically suspected, irrespective of the presence or absence of inflammatory CSF changes.


[Personal recollection of episodes devoted to my study of neuropathology].
I graduated from Kyoto University Faculty of Medicine in 1952. Following neurological residency training, I received neuropathological training at Montefiore Hospital under Dr. Zimmerman since 1956. During 1959-65, on the recommendation from Dr. Zimmerman and Dr. LT Kurlands, I was engaged in Guam project of NIH, as a visiting scientist, investigating ALS and parkinsonism-dementia complex, endemic fatal neurological disorders among the native Chamorro population. In 1965 I was appointed as head of the Division of Neuropathology at Montefiore Medical Center. I have been Professor of Pathology at Albert Einstein College of Medicine since 1971, Professor in the Dominick Purpura Department of Neuroscience at Albert Einstein College of Medicine since 1974, and The Harry M. Zimmerman Professor of Neuropathology, Montefiore Medical Center 1995. For over four decades, with the late Dr. Zimmerman, I have been host to 40 Japanese neurologists who have come to Montefiore for training in Neuropathology. Over 700 papers, 20 books have been published in our laboratory. Personal recollections of selected episodes devoted to study of neuropathology are described in this communication. These include fine structural investigation of brain edema demonstrating electron dense hematogenous edema fluid spreading expanding extracellular space in white matter, application of model of unrolled myelin sheath for elucidate structural alteration of myelin, the independent development of the pre- and postsynaptic terminals, study of SOD1 positive Lewy body-like inclusion in familial ALS and Hirano body.


Amphibian skin-associated Pigmentiphaga: Genome sequence and occurrence across geography and hosts.
The bacterial communities colonizing amphibian skin have been intensively studied due to their interactions with pathogenic chytrid fungi that are causing drastic amphibian population declines. Bacteria of the family Alcaligenaceae, and more specifically of the genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. Here we analyze their occurrence in a previously assembled global skin microbiome dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total number of reads in this global dataset. They were mostly found in unrelated arboreal frogs from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a clade within Pigmentiphaga not assignable to any type strains of the five described species of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome sequence of this bacterium which, based on 16S sequences of >97% similarity, has previously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with environmental information processing and cellular processes. Furthermore, it has a biosynthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosynthetic genes can be found, but clusters for β-lactones present in other comparative Pigmentiphaga genomes are lacking.


Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.
In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These "phasiRNAs" are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.


Minimal camera networks for 3D image based modeling of cultural heritage objects.
3D modeling of cultural heritage objects like artifacts, statues and buildings is nowadays an important tool for virtual museums, preservation and restoration. In this paper, we introduce a method to automatically design a minimal imaging network for the 3D modeling of cultural heritage objects. This becomes important for reducing the image capture time and processing when documenting large and complex sites. Moreover, such a minimal camera network design is desirable for imaging non-digitally documented artifacts in museums and other archeological sites to avoid disturbing the visitors for a long time and/or moving delicate precious objects to complete the documentation task. The developed method is tested on the Iraqi famous statue "Lamassu". Lamassu is a human-headed winged bull of over 4.25 m in height from the era of Ashurnasirpal II (883-859 BC). Close-range photogrammetry is used for the 3D modeling task where a dense ordered imaging network of 45 high resolution images were captured around Lamassu with an object sample distance of 1 mm. These images constitute a dense network and the aim of our study was to apply our method to reduce the number of images for the 3D modeling and at the same time preserve pre-defined point accuracy. Temporary control points were fixed evenly on the body of Lamassu and measured by using a total station for the external validation and scaling purpose. Two network filtering methods are implemented and three different software packages are used to investigate the efficiency of the image orientation and modeling of the statue in the filtered (reduced) image networks. Internal and external validation results prove that minimal image networks can provide highly accurate records and efficiency in terms of visualization, completeness, processing time (>60% reduction) and the final accuracy of 1 mm.


Residential status and birth outcomes: is the rural/urban distinction adequate?
In studies comparing the birth outcomes of rural and urban women, residency status is frequently defined dichotomously as either rural or urban. Since residency status appears to be a continuum, however, the usefulness of other categorization systems needs to be explored. The purpose of this study was to compare birth outcomes using a three-level variable for residency status (rural, rural adjacent to urban, and urban). The study population was comprised of women who delivered by cesarean section over an 18-month period (N = 263) at a tertiary care hospital. Data were collected from patient charts, interviews, and the hospital information system. Residency status was determined by county of residence. Birth outcomes examined included gestational age, birthweight, Apgar scores, maternal complications, length of hospital stay, and costs of hospital care. Rural women had worse birth outcomes overall and traveled the greatest distance for delivery. Rural-adjacent women had the best birth outcomes of the three groups, yet were the youngest, least educated, least likely to be married, and the least likely to be privately insured. By using a nondichotomous three-level variable for residency status, two distinct groups of rural women were identified whose maternal health care needs may differ from each other.


Increased atherogenic lipoproteins are associated with cognitive impairment: effects of statins and subclinical atherosclerosis.
Hypercholesterolemia increases the risk for dementia. Some studies suggest that statins may protect cognition, but findings are conflicting. Unmeasured confounders, including high-density lipoprotein (HDL) cholesterol or subclinical atherosclerosis, may have influenced prior study outcomes. In older adults participating in a population-based cohort study (n=1711, aged 65 to 97 y), we investigated the relationships of total and HDL cholesterol levels, statin use, and carotid intima-media thickness with the prevalence of cognitive impairment. In adjusted models, participants in the highest quartile of non-HDL (total-HDL) cholesterol had an increased odds of cognitive impairment compared with those in the lowest quartile [odds ratio (OR): 2.06, 95% confidence interval (CI): 1.07-3.98]. Statin use was associated with lower odds of cognitive impairment in unadjusted models (OR: 0.57, 95% CI: 0.36-0.89), but this relationship was not significant after adjusting for vascular and lifestyle factors (OR: 0.84, 95% CI: 0.47-1.49). In this analysis of older adults, increased atherogenic lipoproteins were associated with impaired cognition. Statin use was related to many factors that both negatively and positively affect cognition, but was not associated with better cognitive function. These results suggest that confounding by indication may explain the contradictory findings in studies assessing the association of statins with cognition. Randomized-controlled clinical trials and longitudinal studies are necessary to determine if statins protect against cognitive decline.


Twisted π-system chromophores for all-optical switching.
Molecular chromophores with twisted π-electron systems have been shown to possess unprecedented values of the quadratic hyperpolarizability, β, with very large real parts and much smaller imaginary parts. We report here an experimental and theoretical study which shows that these twisted chromophores also possess very large values of the real part of the cubic hyperpolarizability, γ, which is responsible for nonlinear refraction. Thus, for the two-ring twisted chromophore TMC-2 at 775 nm, relatively close to one-photon resonance, n(2) extrapolated to neat substance is large and positive (1.87 × 10(-13) cm(2)/W), leading to self-focusing. Furthermore, the third-order response includes a remarkably low two-photon absorption coefficient, which means minimal nonlinear optical losses: the T factor, α(2)λ/n(2), is 0.308. These characteristics are attributed to closely spaced singlet biradical and zwitterionic states and offer promise for applications in all-optical switching.


Drosophila toll pathway: the new model.
In Drosophila, recognition of microbe-specific molecules (such as bacterial peptidoglycans) activates serine protease cascades that converge to activate the Toll pathway. Recent data show that the serine protease Grass, which is activated downstream of pattern recognition receptors and was initially thought to be a component only of the Gram-positive bacteria-induced signaling cascade, is also required for the induction of the Toll pathway after fungal infection. Persephone, a serine protease known to be specifically activated by fungal proteases, was also found to be required for sensing Gram-positive bacterial proteases. Thus, Persephone serves as a sensor for microbial activities from both fungi and Gram-positive bacteria. With these new discoveries, a new model has been proposed for activation of the Drosophila Toll pathway.


Temporal Differences in Utilization of Intracytoplasmic Sperm Injection Among U.S. Regions.
To analyze changes in intracytoplasmic sperm injection (ICSI) utilization, indications, and outcomes across U.S. regions. We conducted a retrospective cohort study. Data sets for 2000-2014 were obtained from the Centers for Disease Control and Prevention. Clinics with 100 or greater fresh, nondonor cycles were grouped by 10 nationally recognized Department of Health and Human Services (DHHS) regions and were compared for use of ICSI, frequency of male factor infertility, preimplantation genetic therapies, pregnancy, and live birth rates per cycle among fresh in vitro fertilization cycles in women younger than 35 years of age. Nationwide ICSI utilization increased 52% (46.3±6.1% to 70.0±7.1%) from 2000 to 2014, whereas pregnancy and live birth rates per cycle modestly increased by 8.5% (39.2±3.8% to 42.5±2.5%) and 7.6% (34.4±3.6% to 37±2.6%), respectively, showing a positive correlation (r=0.78, P<.001; r=0.76, P=.001) with ICSI rates per clinic. All DHHS Services regions demonstrated increases in ICSI utilization over time, although the magnitude of increase varied in different regions. Regions also had similarities in trends for pregnancy and live birth rates per cycle in women younger than 35 years. There was no correlation between male factor and ICSI rates per clinic from 2000 to 2010 (r=0.32, P=.33) or 2011 to 2014 (r=0.85, P=.068). From 2007 to 2014, ICSI and preimplantation genetic testing did not demonstrate a strong correlation (r=0.68, P=.062). From 2000 to 2014, ICSI rates per clinic significantly varied among geographic regions. Increased use of ICSI did not correlate with an increase in male factor diagnoses. These findings suggest that ICSI may be overused, because its use is not accompanied by proportionate increases in medical indications or effectiveness.


HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes.
We have previously shown that HIV-1-infected children develop broader and more potent neutralizing antibody responses than adults. This study aimed to determine the antibody specificities in 16 HIV-1 subtype C-infected children who displayed exceptional neutralization breadth on a 22-multisubtype virus panel. All children were antiretroviral treatment (ART) naive with normal CD4 counts despite being infected for a median of 10.1 years with high viral loads. The specificity of broadly neutralizing antibodies (bNAbs) was determined using epitope-ablating mutants, chimeric constructs, and depletion or inhibition of activity with peptides and glycoproteins. We found that bNAbs in children largely targeted previously defined epitopes, including the V2-glycan, V3-glycan, CD4bs, and gp120-gp41 interface. Remarkably, 63% of children had antibodies targeting 2 or 3 and, in one case, 4 of these bNAb epitopes. Longitudinal analysis of plasma from a mother-child pair over 9 years showed that while they both had similar neutralization profiles, the antibody specificities differed. The mother developed antibodies targeting the V2-glycan and CD4bs, whereas bNAb specificities in the child could not be mapped until 6 years, when a minor V2-glycan response appeared. The child also developed high-titer membrane-proximal external region (MPER) binding antibodies not seen in the mother, although these were not a major bNAb specificity. Overall, exceptional neutralization breadth in this group of children may be the result of extended exposure to high antigenic load in the context of an intact immune system, which allowed for the activation of multiple B cell lineages and the generation of polyclonal responses targeting several bNAb epitopes.IMPORTANCE An HIV vaccine is likely to require bNAbs, which have been shown to prevent HIV acquisition in nonhuman primates. Recent evidence suggests that HIV-infected children are inherently better at generating bNAbs than adults. Here, we show that exceptional neutralization breadth in a group of viremic HIV-1 subtype C-infected children was due to the presence of polyclonal bNAb responses. These bNAbs targeted multiple epitopes on the HIV envelope glycoprotein previously defined in adult infection, suggesting that the immature immune system recognizes HIV antigens similarly. Since elicitation of a polyclonal bNAb response is the basis of next-generation HIV envelope vaccines, further studies of how bNAb lineages are stimulated in children is warranted. Furthermore, our findings suggest that children may respond particularly well to vaccines designed to elicit antibodies to multiple bNAb epitopes.


Clinical features of gastroduodenal injury associated with long-term low-dose aspirin therapy.
Low-dose aspirin (LDA) is clinically used for the prevention of cardiovascular and cerebrovascular events with the advent of an aging society. On the other hand, a very low dose of aspirin (10 mg daily) decreases the gastric mucosal prostaglandin levels and causes significant gastric mucosal damage. The incidence of LDA-induced gastrointestinal mucosal injury and bleeding has increased. It has been noticed that the incidence of LDA-induced gastrointestinal hemorrhage has increased more than that of non-aspirin non-steroidal anti-inflammatory drug (NSAID)-induced lesions. The pathogenesis related to inhibition of cyclooxygenase (COX)-1 includes reduced mucosal flow, reduced mucus and bicarbonate secretion, and impaired platelet aggregation. The pathogenesis related to inhibition of COX-2 involves reduced angiogenesis and increased leukocyte adherence. The pathogenic mechanisms related to direct epithelial damage are acid back diffusion and impaired platelet aggregation. The factors associated with an increased risk of upper gastrointestinal (GI) complications in subjects taking LDA are aspirin dose, history of ulcer or upper GI bleeding, age > 70 years, concomitant use of non-aspirin NSAIDs including COX-2-selective NSAIDs, and Helicobacter pylori (H. pylori) infection. Moreover, no significant differences have been found between ulcer and non-ulcer groups in the frequency and severity of symptoms such as nausea, acid regurgitation, heartburn, and bloating. It has been shown that the ratios of ulcers located in the body, fundus and cardia are significantly higher in bleeding patients than the ratio of gastroduodenal ulcers in patients taking LDA. Proton pump inhibitors reduce the risk of developing gastric and duodenal ulcers. In contrast to NSAID-induced gastrointestinal ulcers, a well-tolerated histamine H2-receptor antagonist is reportedly effective in prevention of LDA-induced gastrointestinal ulcers. The eradication of H. pylori is equivalent to treatment with omeprazole in preventing recurrent bleeding. Continuous aspirin therapy for patients with gastrointestinal bleeding may increase the risk of recurrent bleeding but potentially reduces the mortality rates, as stopping aspirin therapy is associated with higher mortality rates. It is very important to prevent LDA-induced gastroduodenal ulcer complications including bleeding, and every effort should be exercised to prevent the bleeding complications.


Phagocytosis of inhaled plutonium oxide-239pu particles by pulmonary macrophages.
Pulmonary macrophages and plutonium particles were removed by washing the lungs of rats that had inhaled plutonium oxide-(239)Pu. A significant amount of plutonium was found in multiple washings of the same lung. The removal of toxic particles by washing is of potential therapeutic value. Particles were phagocytized by macrophages during the first 3 hours and retained within these cells for up to 25 days. Nearly all particles in washings were found in macrophages after the second day. The percent of macrophages with engulfed particles increased with increasing amounts of plutonium deposited in the lungs. The ability of pulmonary macrophages to rapidly phagocytize and retain plutonium particles deposited in the lungs has been shown.


Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.
Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.


Rab13 Is Involved in the Entry Step of Hepatitis C Virus Infection.
Membrane transport probably participates in the lifecycle of hepatitis C virus (HCV). Rab proteins are essential host factors for HCV RNA replication, but these proteins' roles in other steps of the HCV lifecycle are not clear. The tight junction (TJ) plays a key role in HCV infection. Rab13 regulates the endocytic recycling of the TJ-associated proteins. Here we investigated whether Rab13 is involved in the HCV entry step. We used HuH-7-derived RSc cells and Li23-derived D7 cells. To evaluate the effect of Rab13 in HCV infection, we transfected the cells with siRNA targeting Rab13 before HCV infection. The down-regulation of Rab13 inhibited HCV infection. The D7 cells had showed a greater inhibitory effect against HCV infection compared to that in the RSc cells by Rab13 knockdown. Next, to evaluate the effect of Rab13 after infection, we inoculated the cells with HCV before transfection of the siRNA. The down-regulation of Rab13 did not show any effects after HCV infection. We further examined whether Rab13 would influence HCV RNA replication by using HCV replicon-harboring cells. The results revealed that Rab13 did not affect the step of HCV RNA replication. These results suggest that Rab13 plays an important role in the step of HCV entry.


50-GHz-spaced comb of high-dimensional frequency-bin entangled photons from an on-chip silicon nitride microresonator.
Quantum frequency combs from chip-scale integrated sources are promising candidates for scalable and robust quantum information processing (QIP). However, to use these quantum combs for frequency domain QIP, demonstration of entanglement in the frequency basis, showing that the entangled photons are in a coherent superposition of multiple frequency bins, is required. We present a verification of qubit and qutrit frequency-bin entanglement using an on-chip quantum frequency comb with 40 mode pairs, through a two-photon interference measurement that is based on electro-optic phase modulation. Our demonstrations provide an important contribution in establishing integrated optical microresonators as a source for high-dimensional frequency-bin encoded quantum computing, as well as dense quantum key distribution.


Mesenchymal Stem Cells and Their Clinical Applications in Osteoarthritis.
Osteoarthritis is a chronic degenerative joint disorder characterized by articular cartilage destruction and osteophyte formation. Chondrocytes in the matrix have a relatively slow turnover rate, and the tissue itself lacks a blood supply to support repair and remodeling. Researchers have evaluated the effectiveness of stem cell therapy and tissue engineering for treating osteoarthritis. All sources of stem cells, including embryonic, induced pluripotent, fetal, and adult stem cells, have potential use in stem cell therapy, which provides a permanent biological solution. Mesenchymal stem cells (MSCs) isolated from bone marrow, adipose tissue, and umbilical cord show considerable promise for use in cartilage repair. MSCs can be sourced from any or all joint tissues and can modulate the immune response. Additionally, MSCs can directly differentiate into chondrocytes under appropriate signal transduction. They also have immunosuppressive and anti-inflammatory paracrine effects. This article reviews the current clinical applications of MSCs and future directions of research in osteoarthritis.


Hormonal and dietary modulation of mammary carcinogenesis in mouse mammary tumor virus-c-erbB-2 transgenic mice.
Exogenous and dietary estrogens have been associated with modification of breast cancer risk. Mammary cancer model systems can be used to explore interactions between specific transgenes, and hormonal and dietary factors. Transgenic mice bearing the rat wild-type erbB-2 gene were used to study the effects of short-term hormonal exposure [17beta-estradiol (E2) or tamoxifen] or a soy meal diet on mammary carcinogenesis. In mice fed a casein diet, mammary tumors developed at an earlier age after short-term E2 exposure during the early reproductive period. The median mammary tumor latency was shortest (29 weeks) for the high-dose estrogen as compared with the lowest dose of E2 treated or placebo control mice (33 and 37 weeks, respectively). The timing of short-term E2 exposure was also important, with the most significant changes observed in mice exposed to E2 between 8 and 18 weeks of age. E2 exposure was associated with the subsequent development of more aggressive tumors as determined by histologic grade, multifocal tumor development, stromal invasion, and pulmonary metastasis. In contrast, short-term tamoxifen-exposed mice generally failed to develop mammary tumors by 60 weeks of age. Mice fed a soy meal diet developed mammary tumors at a later age than casein-fed animals treated with E2 or placebo, whereas no differences were observed by diet for the tamoxifen-treated mice. Mammary tumor prevention was >80% in tamoxifen-treated mice on either diet. Novel histologic tumor types were identified, suggesting greater phenotypic diversity than described previously. Benign mammary gland morphogenesis was also significantly altered by short-term hormonal exposure or dietary factors, consistent with the modification of mammary tumor risk in specific treatment groups. Estrogenic modulation of the mammary tumor phenotype in wild-type erbB-2 transgenic mice was observed. Histologic tumor types and clinical aggressivity not reported previously in this transgenic model were noted, suggesting greater biologic heterogeneity than reported previously. In addition, dietary phytoestrogens modified mammary development and tumor latency, suggesting a need for greater stringency in dietary assignment for transgenic mouse models of mammary neoplasia.


Effect of the Anti-retroviral Drugs Efavirenz, Tenofovir and Emtricitabine on Endothelial Cell Function: Role of PARP.
Highly active anti-retroviral therapy has proved successful in reducing morbidity and mortality associated with HIV infection though it has been linked to increased risk of cardiovascular disease. To date, the direct effects of the anti-retroviral drugs Efavirenz, Tenofovir and Emtricitabine on the vasculature relaxant response have not been elucidated, which impaired may predispose individuals to cardiovascular disease. Increased cellular oxidative stress and overactivation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) have been identified as central mediators of vascular dysfunction. The aim of this study was to investigate whether exposure to Efavirenz, Tenofovir or Emtricitabine directly causes endothelial cell dysfunction via overactivation of PARP. Exposure of ex vivo male rat aortic rings or in vitro endothelial cells to Efavirenz but not Tenofovir or Emtricitabine impaired the acetylcholine-mediated relaxant response, increased cellular oxidative stress and PARP activity, decreased cell viability and increased apoptosis and necrosis. Pharmacological inhibition of PARP protected against the Efavirenz-mediated impairment of vascular relaxation and endothelial cell dysfunction. Oestrogen exposure also protected against the Efavirenz-mediated inhibition of the vascular relaxant response, cell dysfunction and increased PARP activation. In conclusion, Efavirenz directly impairs endothelial cell function, which may account for the increased risk of developing cardiovascular complications with anti-retroviral therapy.


Pediatric phase I trial and pharmacokinetic study of vorinostat: a Children's Oncology Group phase I consortium report.
The purpose of this study was to determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLT), and pharmacokinetics of vorinostat administered as a single agent and in combination 13-cis retinoic acid (13cRA) in children with refractory solid tumors; to evaluate the tolerability of the solid tumor MTD in children with refractory leukemias; and to characterize the pharmacokinetics of a vorinostat suspension in children. Vorinostat was administered orally daily starting at 180 mg/m(2)/d with escalations planned in 30% increments. Pharmacokinetic studies were performed with the initial dose. Acetyl-histone (H3) accumulation was assessed by Western blotting of peripheral blood mononuclear cells (PBMC). Sixty-four patients were enrolled on this multipart trial. In patients with solid tumors, the MTD was 230 mg/m(2)/d with dose-limiting neutropenia, thrombocytopenia, and hypokalemia at 300 mg/m(2)/d. DLTs observed with the combination of 13cRA and vorinostat included thrombocytopenia, neutropenia, anorexia, and hypertriglyceridemia, resulting in a MTD of vorinostat 180 mg/m(2)/d 4 times per week and 13cRA 80 mg/m(2)/dose twice per day, days 1 through 14 every 28 days. Wide interpatient variability was noted in vorinostat disposition, with area under the concentration-time curves at 230 mg/m(2)/d for the capsule (range, 1,415 to 9,291 ng/mL x hr) and oral suspension (range, 1,186 to 4,780 ng/mL x hr). Significant accumulation of acetylated H3 histone in PBMC was observed after administration of vorinostat, particularly at higher doses. One patient with neuroblastoma experienced a complete response to the combination. In children with recurrent solid tumors, vorinostat is well-tolerated at 230 mg/m(2)/d, with a modest dose reduction being required when combining vorinostat with 13cRA. Drug disposition is similar to that observed in adults.


Hindrance for patient participation in nursing care.
THE STUDY'S RATIONALE: Patients' influence in health care through participation, freedom of choice and information, is laid down in laws, national and local directives. In nursing care situations, the degree to which a patient participates depends on the nursing staff. Accordingly, hindrances for patient's participation during nursing care is an important question for the nursing profession. The aim was to focus on Swedish Registered Nurses opinion of hindrances for patient participation in nursing care and to uncover the informants' perspectives in depth. The study was limited to inpatient somatic care and has a qualitative approach. Data were collected through seven focus group interviews with 31 Registered Nurses from five hospitals. An analysis of the tape-recorded interview material was made, combining elements of content analysis with aspects of the Grounded Theory approach. The ethics of scientific work was followed. The participants gave informed consent. Verbal and written information was given as a guarantee that all information would be treated confidentially outside the focus group. Formal approval by ethical committee was not required according to national and local directives. Hindrance for patient participation in nursing care comprised three themes: Competence, Influence of significant others and Organization and work environment, and their seven underlying subthemes. The study clarified factors, which individually or combined may be hindrance for patient participation in nursing practice. Professional nurses must be able to find a balance for their patients' participation in nursing care activities through identification and coping with the hindrances. The three themes and seven subthemes here identified, can be used in patient care and its' evaluation, like also quality assurance of care and work organization and in nursing education. For further development replication studies are needed, like additional studies of patients and significant others.


Muscle damage and muscle remodeling: no pain, no gain?
Skeletal muscle is a dynamic tissue that responds adaptively to both the nature and intensity of muscle use. This phenotypic plasticity ensures that muscle structure is linked to patterns of muscle use throughout the lifetime of an animal. The cascade of events that result in muscle restructuring - for example, in response to resistance exercise training - is often thought to be initiated by muscle damage. We designed this study to test the hypothesis that symptomatic (i.e. detectable) damage is a necessary precursor for muscle remodeling. Subjects were divided into two experimental populations: pre-trained (PT) and naive (NA). Demonstrable muscle damage was avoided in the PT group by a three-week gradual 'ramp-up' protocol. By contrast, the NA group was subjected to an initial damaging bout of exercise. Both groups participated in an eight-week high-force eccentric-cycle ergometry program (20 min, three times per week) designed to equate the total work done during training between the groups. The NA group experienced signs of damage, absent in the PT group, as indicated by greater than five times higher levels of plasma creatine kinase (CK) and self-reporting of initial perceived soreness and exertion, yet muscle size and strength gains were not different for the two groups. RT-PCR analysis revealed similar increases in levels of the growth factor IGF-1Ea mRNA in both groups. Likewise, the significant (P<0.01) increases in mean cross-sectional area (and total muscle volume) were equal in both groups. Finally, strength increases were identical for both groups (PT=25% and NA=26% improvement). The results of this study suggest that muscle rebuilding - for example, hypertrophy - can be initiated independent of any discernible damage to the muscle.


Why are male social relationships complex in the Doubtful Sound bottlenose dolphin population?
Access to oestrus females tends to be the main driver of male sociality. This factor can lead to complex behavioural interactions between males and groups of males. Male bottlenose dolphins may form alliances to consort females and to compete with other males. In some populations these alliances may form temporary coalitions when competing for females. I examined the role of dyadic and group interactions in the association patterns of male bottlenose dolphins in Doubtful Sound, New Zealand. There is no apparent mating competition in this population and no consortship has been observed, yet agonistic interactions between males occur regularly. By comparing the network of male interactions in several social dimensions (affiliative, agonistic, and associative) I show that while agonistic interactions relate to dyadic association patterns, affiliative interactions seem to relate to group association patterns. Some evidence suggests that groups of males also formed temporary coalitions during agonistic interactions. While different groups of males had similar relationships with non-oestrus females, the time they spent with oestrus females and mothers of newborns differed greatly. After considering several hypotheses, I propose that the evolution of these complex relationships was driven by sexual competition probably to out-compete other males for female choice.


Pollution load modelling in sewer systems: an approach of combining long term online sensor data with multi-objective auto-calibration schemes.
Pollutant load modelling for sewer systems is state-of-the-art, especially for the estimation of discharged pollutant loads and development of sewer management strategies. However, conventionally obtained calibration data sets are often not exhaustive and have significant drawbacks. In the Graz West catchment area (Graz, Austria), continuous high-resolution long-term online measurements for discharge and pollutant concentration have been carried out since 2002.In this paper, the application of single- and multi-objective auto-calibration schemes based on evolution strategies for a deterministic hydrological pollutant load model will be discussed. Three approaches for pollutant load modelling are examined and compared: using a constant storm weather concentration and two surface accumulation-wash-off approaches with basic respectively extended wash-off equations. It is shown that the applied auto-calibration method leads to very satisfying results for both the calibration and the validation data set, and also for the dry and the storm weather runoff. Results from multi-objective calibration show better robustness in validation events than single-objective calibration. The build-up wash-off approach using the basic wash-off equation gives the best correlations between measured data and simulation results.


Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) reveals a requirement for MAPK signalling in mouse sex determination.
Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans.


Bacterial alkaline proteases: molecular approaches and industrial applications.
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN' and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.


Acetaminophen pharmacokinetics in geese.
To evaluate the pharmacokinetics of acetaminophen (APAP) after single-dose IV and PO in the goose; to quantify APAP and its main metabolites in goose muscle, heart, lung, liver, and kidney; and to perform a histopathologic evaluation of goose stomach, duodenum, liver, and kidney tissues for potential signs of toxicity. 24 geese. Geese were randomly divided into 3 groups (n = 8). Group I received APAP (10 mg/kg) IV, and groups II and III received the same dose PO. Groups I and II were used for the pharmacokinetic assessment, and group III was used for the residue analysis and histopathologic evaluation. APAP and its metabolites were quantified in plasma and tissues by ultra-high-performance liquid chromatography-tandem mass spectrometry, and the pharmacokinetic analysis was performed using a noncompartmental approach. APAP plasma concentrations were lower than those of the metabolites in similar selected time points after both treatments. After IV treatment, the APAP area under the curve value was statistically higher than that after PO administration, resulting in an oral bioavailability of 46%. In contrast, the area under the curve of the metabolites following PO administration was statistically higher than those found after IV administration. Tissue residues of APAP were highest in the liver, with an accumulation index > 1. Fatty degeneration of hepatocytes was observed 24 hours after administration of APAP. In geese, treatment by PO administration of APAP shows incomplete absorption and a slight accumulation in lung and liver. Tissue alterations occurred in the liver at 24 hours, while no signs of toxicity were found in the other tested organs.


Preliminary evaluation of local drug delivery of amphotericin B and in vivo degradation of chitosan and polyethylene glycol blended sponges.
This research investigated the combination of polyethylene glycol with chitosan in point-of-care loaded sponges made by one or two lyophilizations for adjunctive local antifungal delivery in musculoskeletal wounds. Blended and control chitosan sponges were evaluated in vitro for antifungal release and activity, degradation, cytocompatibility, and characterized for spectroscopic, crystallinity, thermal, and morphologic material properties. In vivo biocompatibility and degradation of sponges were also evaluated in a rat intramuscular pouch model 4 and 10 days after implantation. Blended sponges released amphotericin B active against Candida albicans (>0.25 µg/mL) over 72 h and did not elicit cytotoxicity response of fibroblasts. Blended sponges exhibited decreases in surface roughness, decreased thermal decomposition temperatures, as well as small Fourier transform infrared spectroscopy and crystallinity differences, compared with chitosan-only sponges. Three of the four blended sponge formulations exhibited 31%-94% increases in in vitro degradation from the chitosan sponges after 10 days, but did not demonstrate the same increase in in vivo degradation. Low inflammatory in vivo tissue response to blended and chitosan-only sponges was similar over 10 days. These results demonstrated that adding polyethylene glycol to chitosan sponges does improve local antifungal release, cytocompatibility, and in vitro degradation, but does not increase in vivo degradation.


Dual roles of calpain in facilitating Coxsackievirus B3 replication and prompting inflammation in acute myocarditis.
Viral myocarditis (VMC) treatment has long been lacking of effective methods. Our former studies indicated roles of calpain in VMC pathogenesis. This study aimed at verifying the potential of calpain in Coxsackievirus B3 (CVB3)-induced myocarditis treatment. A transgenic mouse overexpressing the endogenous calpain inhibitor, calpastatin, was introduced in the study. VMC mouse model was established via intraperitoneal injection of CVB3 in transgenic and wild mouse respectively. Myocardial injury was assayed histologically (HE staining and pathology grading) and serologically (myocardial damage markers of CK-MB and cTnI). CVB3 replication was observed in vivo and in vitro via the capsid protein VP1 detection or virus titration. Inflammation/fibrotic factors of MPO, perforin, IFNγ, IL17, Smad3 and MMP2 were evaluated using western blot or immunohistology stain. Role of calpain in regulating fibroblast migration was studied in scratch assays. Calpastatin overexpression ameliorated myocardial injury induced by CVB3 infection significantly in transgenic mouse indicated by reduced peripheral CK-MB and cTnI levels and improved histology injury. Comparing with CVB3-infected wild type mouse, the transgenic mouse heart tissue carried lower virus load. The inflammation factors of MPO, perforin, IFNγ and IL17 were down-regulated accompanied with fibrotic agents of Smad3 and MMP2 inhibition. And calpain participated in the migration of fibroblasts in vitro, which further proves its role in regulating fibrosis. Calpain plays dual roles of facilitating CVB3 replication and inflammation promotion. Calpain inhibition in CVB3-induced myocarditis showed significant treatment effect. Calpain might be a novel target for VMC treatment in clinical practices.


Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.
The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.


Hypoplastic left heart syndrome: consensus and controversies in 2007.
Variability in practice can be considered to foster clinical innovation, and allow for individualized therapeutic plans and independence of practitioners. The Institute of Medicine, however, has issued a report suggesting that variability in patterns of practice are "illogical", and should be avoided whenever possible. Perhaps nowhere in the field of congenital cardiac disease is variability in practice more apparent than in the management of hypoplastic left heart syndrome. This review assesses the variability in practice at a large number of centres that manage neonates with hypoplastic left heart syndrome, with an emphasis on practice before, during, and after the first stage of the Norwood sequence of operations. We also suggest changes in future strategies for research. In March, 2007, colleagues were contacted to respond to an internet-based survey using commercially available software (www.surveymonkey.com) to collect responses about the management practices for neonates with "straight-forward" hypoplastic left heart syndrome. No attempt was made to correlate management practices with any measures of outcome, as neither the practices themselves, nor the outcomes of interest, could be externally validated. Data is reported from 52 centers thought to manage over 1000 neonates with hypoplastic left heart syndrome on an annual basis. The first stage of the Norwood sequence was "recommended" to families by approximately five-sixths (86.5%) of the centres. No centre recommended primary cardiac transplantation, a "hybrid" approach, or non-intervention. In 7 centres (14.5%), it was reported that there was discussion of some or all of the above options, but ultimately the families decided upon the appropriate strategy. Most centres preferentially used antegrade cerebral perfusion (54%) in contrast to deep hypothermia with circulatory arrest (24%), albeit that 11% of centres used a combination of these techniques and in 9% the support strategy was based on surgeon preference. The source of flow of blood for the lungs following the first stage of reconstruction was also highly variable. Of the 51 centres that responded to the question, 13 (25.5%) were participating in a multi-centric randomized clinical trial comparing the modified Blalock-Taussig shunt to the conduit placed from the right ventricle to the pulmonary arteries, the so-called "Sano" modification. Of the remaining 38 centres, 18 "usually" placed a conduit from the right ventricle to the pulmonary artery, 14 "usually" placed a modified Blalock-Taussig shunt, and at six centres, the decision was made "based upon the preference of the surgeon and/or the cardiologist". Similarly, significant variability in practice was evident in preoperative management, other surgical strategies, postoperative medical support, monitoring and discharge planning. Other than the randomized clinical trial of shunt type, no other medical or surgical management strategy was currently under investigation in a multi-centric or randomized trial in the centres who responded to the survey. The survey emphasises the extreme variability in our current practices for treatment of children with hypoplastic left heart syndrome. While there are some areas for which there is consensus in management, the majority of our practices are variable between and within centres. These results emphasize that large multicentric trials and registries are necessary to improve care, and to answer important clinical questions, emphasizing the need to shift from analysis of experiences of single centres to multi-centric and multi-disciplinary collaboration.


Trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus: fine structure and immunohistochemical detection of elastin.
Immunohistochemical and ultrastructural methods were used to examine the distribution of elastin and the fine structure of the trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus. The cells and matrix, as well as the overall organization of these components, in larval and adult trabecular cartilage resemble those of adult annular and piston cartilages (Wright and Youson: Am. J. Anat., 167:59-70, 1983) Chondrocytes are similar to those in hyaline cartilage. Lamprin fibrils and matrix granules, but no collagen fibrils, are found in a matrix arranged into pericellular, territorial, and interterritorial zones. Branchial, pericardial, and nasal cartilages differ from trabecular, annular, and piston cartilages in the organization of their matrix and in the structural components of their matrix and perichondria. Furthermore, immunoreactive elastin-like material is present within the perichondria and peripheral matrices of nasal, branchial, and pericardial cartilages in both larval and adult lampreys. Oxytalan, elaunin, and elastic-like fibers are dispersed between collagen fibers in the perichondrium. The matrix contains lamprin fibrils, matrix granules, and a band of amorphous material, which is reminiscent of elastin, in the periphery bordering the perichondrium. The presence of elastic-like fibers and elastin-like material within some lamprey cartilages implies that this protein may have evolved earlier in vertebrate history than has been previously suggested.


Effect of medial arch support on displacement of the myotendinous junction of the gastrocnemius during standing wall stretching.
Controlled laboratory study. To examine the effects of standing wall stretching with and without medial arch support (WMAS versus WOMAS) on the displacement of the myotendinous junction (DMTJ) of the medial gastrocnemius, rearfoot angle, and navicular height in subjects with neutral foot alignment and pes planus. Standing wall stretching is often prescribed to increase ankle dorsiflexion range of motion for sports fitness and rehabilitation. However, the effect of standing wall stretching WMAS on DMTJ is unknown. Fifteen subjects with neutral foot alignment and 15 subjects with pes planus performed standing wall stretching under WMAS and WOMAS conditions. Measurements of DMTJ and rearfoot position were performed using ultrasonography and video imaging. Navicular height was measured using a ruler. Dependent variables were examined with a 2-way mixed-design analysis of variance. The 2 factors were foot type (neutral foot versus pes planus) and stretching condition (WMAS versus WOMAS). There were significant interactions of medial arch support by foot type for DMTJ, rearfoot angle, and navicular drop (P<.001). A post hoc paired t test showed that standing wall stretching in the WMAS condition significantly increased the DMTJ, compared to stretching in the WOMAS condition, in subjects with neutral foot (mean +/- SD, 9.6 +/- 1.6 versus 10.5 +/- 1.6 mm; difference, 0.9 mm; 99% CI: 0.4-1.4 mm) and in those with pes planus (10.0 +/- 1.8 versus 12.7 +/- 2.0 mm; difference, 2.7 mm; 99% CI: 1.9-3.5 mm) (P<.001). When comparing WOMAS and WMAS, the difference in DMTJ (1.8 mm; 99% CI: 0.9-2.7 mm) was significantly greater in subjects with pes planus than in those with neutral foot (P<.001). Standing wall stretching with medial arch support maintained subtalar joint neutral position and increased the length of the gastrocnemius in subjects with pes planus. When prescribing standing wall stretching, clinicians need to emphasize the use of medial arch support to effectively stretch the gastrocnemius in subjects with pes planus.


Risk factors for CMV retinitis among individuals with HIV and low CD4 count in northern Thailand: importance of access to healthcare.
To determine if poor access to healthcare is associated with increased cytomegalovirus (CMV) retinitis risk among patients with HIV with CD4 counts of <100 cells/μL screened in a resource-limited setting. This is a prospective cross-sectional study. Patients with known HIV and a CD4 count of <100 cells/μL attending an HIV clinic in Chiang Mai, Thailand, completed a standardised questionnaire and underwent dilated fundus examination. Participants without CMV retinitis were invited for repeated examinations every 3 months until their CD4 count exceeded 100 cells/μL. The relationship between various potential risk factors and CMV retinitis was assessed with logistic regression. 103 study participants were enrolled. At enrolment, the mean age was 37.5 (95% CI 35.7 to 39.2) years, 61.2% (95% CI 51.6% to 70.7%) were male and the mean CD4 count was 29.5 (95% CI 25.9 to 33.1) cells/μL. 21 eyes from 16 (15.5%) participants were diagnosed with CMV retinitis. In multivariate analyses, CMV retinitis was significantly associated with lower CD4 count (OR 1.42 per 10-cell decrement, 95%CI 1.05 to 1.93), longer travel time to clinic (OR 3.85 for those with >30-min travel time, 95% CI 1.08 to 13.8) and lower income (OR 1.22 per US$50 less income, 95% CI 1.02 to 1.47). CD4 count, low income and longer travel time to clinic were significant risk factors for CMV retinitis among patients with HIV in a resource-limited setting. These results suggest that reducing blindness from CMV retinitis should focus on increasing accessibility of screening examinations to poor and hard-to-reach patients.


The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules.
Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signalling. Here, we report the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit encoded by the pHCl-2 (CG11340) gene, in Drosophila melanogaster We show that pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. We demonstrate that pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules and that loss of pHCl-2 reduces urine production during diuresis. Our data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis.


Use of medical simulation to explore equipment failures and human-machine interactions in anesthesia machine pipeline supply crossover.
High-fidelity medical simulation can be used to explore failure modes of technology and equipment and human-machine interactions. We present the use of an equipment malfunction simulation scenario, oxygen (O(2))/nitrous oxide (N(2)O) pipeline crossover, to probe residents' knowledge and their use of anesthetic equipment in a rapidly escalating crisis. In this descriptive study, 20 third-year anesthesia residents were paired into 10 two-member teams. The scenario involved an Ohmeda Modulus SE 7500 anesthetic machine with a Datex AS/3 monitor that provided vital signs and gas monitoring. Before the scenario started, we switched pipeline connections so that N(2)O entered through the O(2) pipeline and vice versa. Because of the switched pipeline, the auxiliary O(2) flowmeter delivered N(2)O instead of O(2). Two expert, independent raters reviewed videotaped scenarios and recorded the alarms explicitly noted by participants and methods of ventilation. Nine pairs became aware of the low fraction of inspired O(2) (Fio(2)) alarm. Only 3 pairs recognized the high fraction of inspired N(2)O (Fin(2)o) alarm. One group failed to recognize both the low Fio(2) and the high Fin(2)o alarms. Nine groups took 3 or more steps before instigating a definitive route of oxygenation. Seven groups used the auxiliary O(2) flowmeter at some point during the management steps. The fact that so many participants used the auxiliary O(2) flowmeter may expose machine factors and related human-machine interactions during an equipment crisis. Use of the auxiliary O(2) flowmeter as a presumed external source of O(2) contributed to delays in definitive treatment. Many participants also failed to notice the presence of high N(2)O. This may have been, in part, attributable to 2 facts that we uncovered during our video review: (a) the transitory nature of the "high N(2)O" alert, and (b) the dominance of the low Fio(2) alarm, which many chose to mute. We suggest that the use of high-fidelity simulations may be a promising avenue to further examine hypotheses related to failure modes of equipment and possible management response strategies of clinicians.


Physiologic control of IDO competence in splenic dendritic cells.
Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.


Neighborhood characteristics and hypertension.
: The goal of this study was to investigate cross-sectional associations between features of neighborhoods and hypertension and to examine the sensitivity of results to various methods of estimating neighborhood conditions. : We used data from the Multi-Ethnic Study of Atherosclerosis on 2612 individuals 45-85 years of age. Hypertension was defined as systolic blood pressure above 140 mm Hg, diastolic pressure above 90 mm Hg, or use of antihypertensive medications. Neighborhood (census tract) conditions potentially related to hypertension (walking environment, availability of healthy foods, safety, social cohesion) were measured using information from a separate phone survey conducted in the study neighborhoods. For each neighborhood we estimated scale scores by aggregating residents' responses using simple aggregation (crude means) and empirical Bayes estimation (unconditional, conditional, and spatial). These estimates of neighborhood conditions were linked to each study participant based on the census tract of residence. Two-level binomial regression methods were used to estimate adjusted associations between neighborhood conditions and hypertension. : Residents of neighborhoods with better walkability, availability of healthy foods, greater safety, and more social cohesion were less likely to be hypertensive (relative prevalence [95% confidence interval] for 90th vs. 10th percentile of conditional empirical Bayes estimate = 0.75 [0.64-0.88], 0.72 [0.61-0.85], 0.74 [0.63-0.86], and 0.69 [0.57-0.83]), respectively, after adjusting for site, age, sex, income, and education. Associations were attenuated and often disappeared after additional adjustments for race/ethnicity. : Neighborhood walkability, food availability, safety, and social cohesion may be mechanisms that link neighborhoods to hypertension.


Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.
Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.


Correlates of lifetime trauma exposure among pregnant women from Cape Town, South Africa.
A cross-sectional survey of 298 pregnant women from Cape Town, South Africa was conducted to examine socio-demographic, reproductive health, mental health, and relationship correlates of lifetime trauma exposure and whether these correlates vary as a function of age. Overall, 19.8% of participants reported trauma exposure. We found similarities and differences in correlates of trauma exposure among women in emerging adulthood and older women. Prior termination of pregnancy was associated with trauma exposure in both age groups. Difficulties in resolving arguments, lifetime substance use, and a prior sexually transmitted infection were associated with trauma exposure among women in emerging adulthood. In contrast, depression and awareness of substance abuse treatment programmes were associated with trauma exposure among older women. These findings highlight the need for interventions that prevent and treat trauma exposure among vulnerable women. Such interventions should be tailored to address the correlates of trauma exposure in each age group.


Structure-based discovery of dengue virus protease inhibitors.
Dengue virus belongs to the family Flaviviridae and is a major emerging pathogen for which the development of vaccines and antiviral therapy has seen little success. The NS3 viral protease is a potential target for antiviral drugs since it is required for virus replication. The goal of this study was to identify novel dengue virus (type 2; DEN2V) protease inhibitors for eventual development as effective anti-flaviviral drugs. The EUDOC docking program was used to computationally screen a small-molecule library for compounds that dock into the P1 pocket and the catalytic site of the DEN2V NS3 protease domain apo-structure [Murthy, K., Clum, S., Padmanabhan, R., 1999. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. J. Biol. Chem. 274, 5573-5580] and the Bowman-Birk inhibitor-bound structure [Murthy, K., Judge, K., DeLucas, L., Padmanabhan, R., 2000. Crystal structure of dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design. J. Mol. Biol. 301, 759-767]. The top 20 computer-identified hits that demonstrated the most favorable scoring "energies" were selected for in vitro assessment of protease inhibition. Preliminary protease activity assays demonstrated that more than half of the tested compounds were soluble and exhibited in vitro inhibition of the DEN2V protease. Two of these compounds also inhibited viral replication in cell culture experiments, and thus are promising compounds for further development.


Implementation of change in health care in Sweden: a qualitative study of professionals' change responses.
Implementation of evidence-based practices in health care implies change. Understanding health care professionals' change responses may be critical for facilitating implementation to achieve an evidence-based practice in the rapidly changing health care environment. The aim of this study was to investigate health care professionals' responses to organizational and workplace changes that have affected their work. We conducted interviews with 30 health care professionals (physicians, registered nurses and assistant nurses) employed in the Swedish health care system. An inductive approach was applied, using a semi-structured interview guide developed by the authors. We used an analytical framework first published in 1999 to analyze the informants' change responses in which change responses are perceived as a continuum ranging from a strong acceptance of change to strong resistance to change, describing seven forms of change responses along this continuum. Change response is conceptualized as a tridimensional attitude composed of three components: cognitive, affective and intentional/behavioral. Analysis of the data yielded 10 types of change responses, which could be mapped onto 5 of the 7 change response categories in the framework. Participants did not report change responses that corresponded with the two most extreme forms of responses in the framework, i.e., commitment and aggressive resistance. Most of the change responses were classified as either indifference or passive resistance to changes. Involvement in or support for changes occurred when the health care professionals initiated the changes themselves or when the changes featured their active input and when changes were seen as well founded and well communicated. We did not identify any change responses that could not be fitted into the framework. We found the framework to be useful for a nuanced understanding of how people respond to changes. This knowledge of change responses is useful for the management of changes and for efforts to achieve more successful implementation of evidence-based practices in health care.


Evidence for widespread distribution of piscidin antimicrobial peptides in teleost fish.
Antimicrobial peptides (AMPs) are increasingly recognized as a critical component of the host's defense against infection. Several types of AMPs have been recently identified from mucosal tissues or immune cells of a number of teleosts. Among these are the piscidins, which are 22 residue, alpha-helical AMPs that were originally isolated from mast cells of hybrid striped bass Morone saxatilis male x Morone chrysops female. Using an antibody specific for the conserved N-terminal amino acid sequence of piscidin 1, we used immunohistochemistry to probe skin, gill, and gastrointestinal tract of 39 teleosts representing 7 different orders. Nine fish species were piscidin-positive, with all of these species being in the Perciformes, the largest and most evolutionarily advanced order of teleosts. Piscidin-positive cells were identified in species belonging to the families Moronidae, Serranidae, Sciaenidae, Siganidae and Belontidae. Immunopositive cells were usually most consistent with mast cells, although in some species, the granule appearance and tinctorial properties diverged somewhat from those of a typical piscine mast cell. In addition, rodlet cells were piscidin-positive in one member of the family Cichlidae; to our knowledge, it is the first time that a host-associated chemical biomarker has been identified in rodlet cells. Our data suggest that piscidins are present in many evolutionarily advanced teleosts. Piscidin-immunoreactive cells were most common at sites of pathogen entry, including the skin, gill and gastrointestinal tract. These results strongly suggest that piscidins are a widespread and important component of many fishes' defense against disease.


Tandem Duplications and the Limits of Natural Selection in Drosophila yakuba and Drosophila simulans.
Tandem duplications are an essential source of genetic novelty, and their variation in natural populations is expected to influence adaptive walks. Here, we describe evolutionary impacts of recently-derived, segregating tandem duplications in Drosophila yakuba and Drosophila simulans. We observe an excess of duplicated genes involved in defense against pathogens, insecticide resistance, chorion development, cuticular peptides, and lipases or endopeptidases associated with the accessory glands across both species. The observed agreement is greater than expectations on chance alone, suggesting large amounts of convergence across functional categories. We document evidence of widespread selection on the D. simulans X, suggesting adaptation through duplication is common on the X. Despite the evidence for positive selection, duplicates display an excess of low frequency variants consistent with largely detrimental impacts, limiting the variation that can effectively facilitate adaptation. Standing variation for tandem duplications spans less than 25% of the genome in D. yakuba and D. simulans, indicating that evolution will be strictly limited by mutation, even in organisms with large population sizes. Effective whole gene duplication rates are low at 1.17 × 10-9 per gene per generation in D. yakuba and 6.03 × 10-10 per gene per generation in D. simulans, suggesting long wait times for new mutations on the order of thousands of years for the establishment of sweeps. Hence, in cases where adaptation depends on individual tandem duplications, evolution will be severely limited by mutation. We observe low levels of parallel recruitment of the same duplicated gene in different species, suggesting that the span of standing variation will define evolutionary outcomes in spite of convergence across gene ontologies consistent with rapidly evolving phenotypes.


From health advice to taboo: community perspectives on the treatment of sleeping sickness in the Democratic Republic of Congo, a qualitative study.
Socio-cultural and economic factors constitute real barriers for uptake of screening and treatment of Human African Trypanosomiasis (HAT) in the Democratic Republic of Congo (DRC). Better understanding and addressing these barriers may enhance the effectiveness of HAT control. We performed a qualitative study consisting of semi-structured interviews and focus group discussions in the Bandundu and Kasaï Oriental provinces, two provinces lagging behind in the HAT elimination effort. Our study population included current and former HAT patients, as well as healthcare providers and program managers of the national HAT control program. All interviews and discussions were voice recorded on a digital device and data were analysed with the ATLAS.ti software. Health workers and community members quoted a number of prohibitions that have to be respected for six months after HAT treatment: no work, no sexual intercourse, no hot food, not walking in the sun. Violating these restrictions is believed to cause serious, and sometimes deadly, complications. These strong prohibitions are well-known by the community and lead some people to avoid HAT screening campaigns, for fear of having to observe such taboos in case of diagnosis. The restrictions originally aimed to mitigate the severe adverse effects of the melarsoprol regimen, but are not evidence-based and became obsolete with the new safer drugs. Correct health information regarding HAT treatment is essential. Health providers should address the perspective of the community in a constant dialogue to keep abreast of unintended transformations of meaning.


Prejudiced interactions: implicit racial bias reduces predictive simulation during joint action with an out-group avatar.
During social interactions people automatically apply stereotypes in order to rapidly categorize others. Racial differences are among the most powerful cues that drive these categorizations and modulate our emotional and cognitive reactivity to others. We investigated whether implicit racial bias may also shape hand kinematics during the execution of realistic joint actions with virtual in- and out-group partners. Caucasian participants were required to perform synchronous imitative or complementary reach-to-grasp movements with avatars that had different skin color (white and black) but showed identical action kinematics. Results demonstrate that stronger visuo-motor interference (indexed here as hand kinematics differences between complementary and imitative actions) emerged: i) when participants were required to predict the partner's action goal in order to on-line adapt their own movements accordingly; ii) during interactions with the in-group partner, indicating the partner's racial membership modulates interactive behaviors. Importantly, the in-group/out-group effect positively correlated with the implicit racial bias of each participant. Thus visuo-motor interference during joint action, likely reflecting predictive embodied simulation of the partner's movements, is affected by cultural inter-individual differences.


Interventions to support the mental health of survivors of modern slavery and human trafficking: A systematic review.
Modern slavery is a term which incorporates a range of exploitative situations that involve the violation of human rights and the subjugation of individuals. It presents a significant public health concern. Post-release, survivors of modern slavery have complex mental health needs. Whilst mental health provision is a component of international and national policy, the delivery of evidence-based support remains a gap in the global anti-slavery response. To identify and synthesise the evidence base for mental health interventions developed and evaluated for use in a post-slavery survivor population. A systematic literature review was undertaken. The review protocol was prospectively registered with PROSPERO and followed the PRISMA guidance in its reporting. A multi-stage search strategy was utilised to retrieve studies. Quality appraisal was undertaken using the QualSyst tool. Due to heterogeneity in study design, a narrative approach to synthesising the findings was undertaken. Nine studies met the final inclusion criteria. The narrative synthesis clustered the studies in three themes: study design and population; type of intervention; and outcomes reported. The included studies focussed on specific subpopulations, namely child soldiering, child labour or sex trafficking. This review has highlighted not only important theory-practice gaps in relation to the provision of evidence-based mental health support but scant evidence limited to specific sub-groups (child soldiering, child labour or sex trafficking). The emphasis placed on PTSD within the interventions tested risks mental health support becoming exclusionary to those with other needs. When assessing intervention efficacy, the complex socio-political context in which survivors exist as well as the increasing emphasis on holistic care, personal recovery and lived experience need to be considered. Taking this into account, the case can be made for the inclusion of a wider range of non-clinical outcomes in the assessment of mental health intervention effectiveness.


ADCY5-related dyskinesia presenting as familial myoclonus-dystonia.
We describe a family with an autosomal dominant familial dyskinesia resembling myoclonus-dystonia associated with a novel missense mutation in ADCY5, found through whole-exome sequencing. A tiered analytical approach was used to analyse whole-exome sequencing data from an affected grandmother-granddaughter pair. Whole-exome sequencing identified 18,000 shared variants, of which 46 were non-synonymous changes not present in a local cohort of control exomes (n = 422). Further filtering based on predicted splicing effect, minor allele frequency in the 1000 Genomes Project and on phylogenetic conservation yielded 13 candidate variants, of which the heterozygous missense mutation c.3086T>G, p. M1029R in ADCY5 most closely matched the observed phenotype. This report illustrates the utility of whole-exome sequencing in cases of undiagnosed movement disorders with clear autosomal dominant inheritance. Moreover, ADCY5 mutations should be considered in cases with apparent myoclonus-dystonia, particularly where SCGE mutations have been excluded. ADCY5-related dyskinesia may manifest variable expressivity within a single family, and affected individuals may be initially diagnosed with differing neurological phenotypes.


Evolution of breast cancer screening in the Medicare population: clinical and economic implications.
Newer approaches to mammography, including digital image acquisition and computer-aided detection (CAD), and adjunct imaging (e.g., magnetic resonance imaging [MRI]) have diffused into clinical practice. The impact of these technologies on screening-related cost and outcomes remains undefined, particularly among older women. Using the Surveillance, Epidemiology, and End Results-Medicare linked database, we constructed two cohorts of women without a history of breast cancer and followed each cohort for 2 years. We compared the use and cost of screening mammography including digital mammography and CAD, adjunct procedures including breast ultrasound, MRI, and biopsy between the period of 2001 and 2002 and the period of 2008 and 2009 using χ(2) and t test. We also assessed the change in breast cancer stage and incidence rates using χ(2) and Poisson regression. All statistical tests were two-sided. There were 137150 women (mean age = 76.0 years) in the early cohort (2001-2002) and 133097 women (mean age = 77.3 years) in the later cohort (2008-2009). The use of digital image acquisition for screening mammography increased from 2.0% in 2001 and 2002 to 29.8% in 2008 and 2009 (P < .001). CAD use increased from 3.2% to 33.1% (P < .001). Average screening-related cost per capita increased from $76 to $112 (P < .001), with annual national fee-for-service Medicare spending increasing from $666 million to $962 million. There was no statistically significant change in detection rates of early-stage tumors (2.45 vs 2.57 per 1000 person-years; P = .41). Although breast cancer screening-related costs increased substantially from 2001 through 2009 among Medicare beneficiaries, a clinically significant change in stage at diagnosis was not observed.


Peptide secondary structure modulates single-walled carbon nanotube fluorescence as a chaperone sensor for nitroaromatics.
A class of peptides from the bombolitin family, not previously identified for nitroaromatic recognition, allows near-infrared fluorescent single-walled carbon nanotubes to transduce specific changes in their conformation. In response to the binding of specific nitroaromatic species, such peptide-nanotube complexes form a virtual "chaperone sensor," which reports modulation of the peptide secondary structure via changes in single-walled carbon nanotubes, near-infrared photoluminescence. A split-channel microscope constructed to image quantized spectral wavelength shifts in real time, in response to nitroaromatic adsorption, results in the first single-nanotube imaging of solvatochromic events. The described indirect detection mechanism, as well as an additional exciton quenching-based optical nitroaromatic detection method, illustrate that functionalization of the carbon nanotube surface can result in completely unique sites for recognition, resolvable at the single-molecule level.


Food insufficiency and physical and mental health in a longitudinal survey of welfare recipients.
Food insufficiency is a significant problem in the United States, and poor African American women with children are at especially high risk. An inadequate household food supply can potentially affect the well-being of household members, but it is difficult to distinguish the effects of food insufficiency from risk factors for poor health that are also common among the food insufficient, such as poverty. We examined food insufficiency and physical and mental health among African American and white women (n = 676) who were welfare recipients in 1997. Controlling for common risk factors, women who reported food insufficiency in both 1997 and 1998 were more likely to report fair or poor health at the later date. Food insufficiency in 1998 was significantly associated with meeting the diagnostic screening criteria for recent major depression. Food insufficiency at both times and in 1998 only was related to women's sense of mastery. These findings add to growing evidence that household food insufficiency is associated with poor physical and mental health.


Characterization of virulence properties in the C. parapsilosis sensu lato species.
The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C. metapsilosis and 18 C. orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C. metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C. orthopsilosis and C. metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C. metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleriamellonella larvae. The mortality rate of G. mellonella larvae infected with C. metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C. orthopsilosis strains. Taken together, our findings demonstrate that C. metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.


The role of luxS in the fire blight pathogen Erwinia amylovora is limited to metabolism and does not involve quorum sensing.
Erwinia amylovora is a gram-negative phytopathogen that causes fire blight of pome fruit and related members of the family Rosaceae. We sequenced the putative autoinducer-2 (AI-2) synthase gene luxS from E. amylovora. Diversity analysis indicated that this gene is extremely conserved among E. amylovora strains. Quorum sensing mediated by LuxS has been implicated in coordinated gene expression, growth, and virulence in other enterobacteria; however, our evidence suggests this is not the function in E. amylovora. Mutational analysis pointed to a role in colonization of apple blossoms, the primary infection court for fire blight, although little if any role in virulence on apple shoots and pear fruit was observed. Expression of key virulence genes hrpL and dspA/E was reduced in mutants of two E. amylovora strains. Stronger effects on gene expression were observed for metabolic genes involved in the activated methyl cycle with mutants having greater levels of expression. No quorum-sensing effect was observed in coculture experiments with wild-type and mutant strains either in vitro or in apple blossoms. Known receptors essential for AI-2 quorum sensing, the LuxPQ sensor kinase or the Lsr ABC-transporter, are absent in E. amylovora, further suggesting a primarily metabolic role for luxS in this bacterium.


Design, Preparation, and Characterization of Zn and Cu Metallopeptides Based On Tetradentate Aminopyridine Ligands Showing Enhanced DNA Cleavage Activity.
The conjugation of redox-active complexes that can function as chemical nucleases to cationic tetrapeptides is pursued in this work in order to explore the expected synergistic effect between these two elements in DNA oxidative cleavage. Coordination complexes of biologically relevant first row metal ions, such as Zn(II) or Cu(II), containing the tetradentate ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane ((Me2)PyTACN) and (2S,2S')-1,1'-bis(pyrid-2-ylmethyl)-2,2'-bipyrrolidine ((S,S')-BPBP) have been linked to a cationic LKKL tetrapeptide sequence. Solid-phase synthesis of the peptide-tetradentate ligand conjugates has been developed, and the preparation and characterization of the corresponding metallotetrapeptides is described. The DNA cleavage activity of Cu and Zn metallopeptides has been evaluated and compared to their metal binding conjugates as well as to the parent complexes and ligands. Very interestingly, the oxidative Cu metallopeptides 1Cu and 2Cu show an enhanced activity compared to the parent complexes, [Cu(PyTACN)](2+) and [Cu(BPBP)](2+), respectively. Under optimized conditions, 1Cu displays an apparent pseudo first-order rate constant (kobs) of ∼0.16 min(-1) with a supercoiled DNA half-life time (t1/2) of ∼4.3 min. On the other hand, kobs for 2Cu has been found to be ∼0.11 min(-1) with t1/2 ≈ 6.4 min. Hence, these results point out that the DNA cleavage activities promoted by the metallopeptides 1Cu and 2Cu render ∼4-fold and ∼23 rate accelerations in comparison with their parent Cu complexes. Additional binding assays and mechanistic studies demonstrate that the enhanced cleavage activities are explained by the presence of the cationic LKKL tetrapeptide sequence, which induces an improved binding affinity to the DNA, thus bringing the metal ion, which is responsible for cleavage, in close proximity.


CHARMM-GUI 10 years for biomolecular modeling and simulation.
CHARMM-GUI, http://www.charmm-gui.org, is a web-based graphical user interface that prepares complex biomolecular systems for molecular simulations. CHARMM-GUI creates input files for a number of programs including CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Since its original development in 2006, CHARMM-GUI has been widely adopted for various purposes and now contains a number of different modules designed to set up a broad range of simulations: (1) PDB Reader & Manipulator, Glycan Reader, and Ligand Reader & Modeler for reading and modifying molecules; (2) Quick MD Simulator, Membrane Builder, Nanodisc Builder, HMMM Builder, Monolayer Builder, Micelle Builder, and Hex Phase Builder for building all-atom simulation systems in various environments; (3) PACE CG Builder and Martini Maker for building coarse-grained simulation systems; (4) DEER Facilitator and MDFF/xMDFF Utilizer for experimentally guided simulations; (5) Implicit Solvent Modeler, PBEQ-Solver, and GCMC/BD Ion Simulator for implicit solvent related calculations; (6) Ligand Binder for ligand solvation and binding free energy simulations; and (7) Drude Prepper for preparation of simulations with the CHARMM Drude polarizable force field. Recently, new modules have been integrated into CHARMM-GUI, such as Glycolipid Modeler for generation of various glycolipid structures, and LPS Modeler for generation of lipopolysaccharide structures from various Gram-negative bacteria. These new features together with existing modules are expected to facilitate advanced molecular modeling and simulation thereby leading to an improved understanding of the structure and dynamics of complex biomolecular systems. Here, we briefly review these capabilities and discuss potential future directions in the CHARMM-GUI development project. © 2016 Wiley Periodicals, Inc.


Towards a definition of life.
This article offers a new definition of life as a "self-contained, self-regulating, self-organizing, self-reproducing, interconnected, open thermodynamic network of component parts which performs work, existing in a complex regime which combines stability and adaptability in the phase transition between order and chaos, as a plant, animal, fungus, or microbe." Open thermodynamic networks, which create and maintain order and are used by all organisms to perform work, import energy from and export entropy into the environment. Intra- and extracellular interconnected networks also confer order. Although life obeys the laws of physics and chemistry, the design of living organisms is not determined by these laws, but by Darwinian selection of the fittest designs. Over a short range of normalized energy consumption, open thermodynamic systems change from deeply ordered to chaotic, and life is found in this phase transition, where a dynamic balance between stability and adaptability allows for homeokinesis. Organisms and cells move within the phase transition with changes in metabolic rate. Seeds, spores and cryo-preserved tissue are well within the ordered regime, while health probably cannot be maintained with displacements into the chaotic regime. Understanding life in these terms may provide new insights into what constitutes health and lead to new theories of disease.